High purity polysaccharide containing hydrophobic group and process for producing it

ABSTRACT

1. A high purity polysaccharide containing hydrophobic group is produced by a process, comprising  
     a first process step of producing an isocyanate group-containing hydrophobic compound, wherein one mole of a hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or of a sterol is reacted with a diisocyanate represented by OCN—R 1 —NCO in which R 1  is a hydrocarbyl of 1-50 carbon atoms,  
     a second process step of producing the polysaccharide containing hydrophobic group composed of the hydrocarbon group of 12-50 carbon atoms or of the steryl group, wherein the isocyanate group-containing hydrophobic compound obtained in the first process step is reacted with one or more polysaccharides, and  
     a purification step in which the reaction product in the second process step is purified using a solvent based on ketone.

FIELD OF THE TECHNIQUE

[0001] The present invention relates to a high purity polysaccharidecontaining hydrophobic group and to a process for producing it.

BACKGROUND OF THE TECHNIQUE

[0002] In water-soluble polymeric substances, there are naturaloccurring polymeric substances, semisynthetic polymeric products andsynthetic polymers. As the natural polymeric substances, for example,carbohydrates, such as starches and marine plant products,mucosubstances, such as gum arabic and the like, and proteins, such asglue and so on. As the semisynthetic polymeric products, there may beenumerated, for example, polymeric cellulose-like substances, such asviscose etc. As the synthetic polymers, there may be exemplifiedpolyvinyl alcohol, polyvinylpyridine and polyglycerol. Some of thepolymeric derivatives having hydrophobic groups derived from thesewater-soluble polymeric substances have now found their applications tomedical materials, such as coating material for coating drug carrierswhich contain drugs. For example, it has been known that, by coating adrug carrier, such as liposome microcapsule, microsphere, O/W emulsionor erythrocyte ghost, with a hydrophobic group-containingpolysaccharide, not only the spontaneous exudation of drug from such adrug carrier is suppressed but also the cell-specific drug transferencerate using such a drug carrier is improved.

[0003] In particular, those compounds in which the water-solublepolymeric substances are polysaccharides and the hydrophobic group issteryl group, namely, polysaccharide-sterol derivatives, have alreadybeen disclosed as a polysaccharide coating material for liposomes(Japanese Patent Kokai Sho 61-69801 A), as a coating material for fatemulsion (Japanese Patent Kokai Sho 63-319046 A) and as a polymericsurfactant to be used on preparation of a polysaccharide-coated emulsion(Japanese Patent Kokai Hei 2-144140 A) and a technique for synthesizingit has been disclosed in Japanese Patent Kokai Sho 61-69801 A.

[0004] It has in recent years been widely accepted that liposome and O/Wemulsion are prospective as drug carrier. It has been reported that thechemical and physical stabilities of a drug carrier of this kind inliving body are improved by coating the drug carrier withpolysaccharide, while thereby a targettropism to a specific cell groupis also revealed {Bull. Chem. Soc. Japan, 62-791 - 796 (1989)}.

[0005] For synthesizing the polysaccharide-cholesterol derivative to beused therefor, a technique has hitherto been employed as described inJapanese Patent Kokai Sho 61-69801 A, which comprises the followingthree process steps, namely,

[0006] reacting a polysaccharide with monochloroacetic acid tosynthesize a carboxymethylated polysaccharide (the first process step),

[0007] reacting the carboxymethylated polysaccharide withethylenediamine to synthesize N-(2-aminoethyl)carbamoylmethlatedpolysaccharide (the second process step) and

[0008] reacting, then, the N-(2-aminoethyl)carbamoylmethylatedpolysaccharide with cholesteryl chloroformate to synthsizeN-{2-(cholesteryloxycarbonylamino)ethyl}carbamoylmethylatedpolysaccharide (the third process step).

[0009] However, this technique disclosed in Japanese Patent Kokai Sho61-69801 suffers from a disadvantage that the carboxyl group of thereactant in the second process step is liable to remain unreacted untilthe end of the process, so that the influence of the negative charge ofsuch remaining carboxyl group on the physicochemical stability,cell-specificity, adaptability and so on of the liposome or the emulsioncoated with the polysaccharide cannot be obviated. A still furtherproblem remains in that this technique requires many process steps.

[0010] In order to resolve these problems, an alternative technique forthe synthesis is proposed in Japanese Patent Kokai Hei 3-292301 A, whichcomprises reacting a diisocyanate with a sterol in the first step tosynthesize a monoisocyanate compound having a steryl group at theα-position at one end of an alkane and an isocyanato group at theω-position of the other end thereof and reacting the monoisocyanatecompound with the polysaccharide in the second step to attain an easyintroduction of steryl group into the polysaccharide.

[0011] However, this technique has disadvantaged in that (1) theby-product formed by the reaction of one mole of the diisocyanatecompound with two moles of the sterol in the first step (in thefollowing, referred to sometimes as the sterol dimer) is not able to beremoved completely by a purification technique by dialysis orreprecipitation using ethanol and the sterol dimer will remain in thefinal product of polysaccharide-sterol derivative as impurity and that(2) the unsubstituted polysaccharide which has not reacted with themonoisocyanate compound (occasionally referred to as unreactedpolysaccharide) in the second step will be present in the final productof polysaccharide-sterol derivative as impurity.

[0012] In using a polysaccharide-sterol derivative for a drug carrier oras a coating material for liposomes, a polysaccharide-sterol derivativeof high purity having fewer content of by-product is to be expected.

[0013] There are detailed reports as to the above-mentionedpolysaccharide-sterol derivatives and as to amphiphilic compositeproducts in which a hydrophobic group other than sterol is bound to awater-soluble polymeric substance other than polysaccharide, such asalkyl diesters of polyethylene glycol {Dojin News No. 85, p 3-11(1997)}.

[0014] However, there is no report up to date as to hydrophobicgroup-containing water-soluble polymeric substance of high purity, dueto existing difficulty in the production of such substance, as mentionedabove.

[0015] The first object of the present invention is to propose a processpermitting production of a high purity polysaccharide containinghydrophobic group with scarce content of impurities, such asunsubstituted polysaccharide and sterol dimer, in an efficient and easymanner.

[0016] The second object of the present invention is to provide a highpurity polysaccharide containing hydrophobic group to be obtained by theproduction process mentioned above.

DISCLOSURE OF THE INVENTION

[0017] The inventors had performed sound researches in respect of theproblems in the prior art give above and reached the discovery that ahigh purity polysaccharide containing hydrophobic group was able to beobtained by employing a combination of technical measures of use of asolvent based on ketone in the reprecipitation process and ofpurification by means of an ultracentrifugation or of purification withan aprotic polar solvent, whereby the present invention has beencompleted. Thus, the present invention consists in the high puritypolysaccharide containing hydrophobic group and in the process forproducing it as given below:

[0018] (1) A process for producing a high purity polysaccharidecontaining hydrophobic group, comprising

[0019] a first process step of producing an isocyanate group-containinghydrophobic compound, wherein one mole of a hydroxyl group-containinghydrocarbon having 12-50 carbon atoms or of a sterol is reacted with adiisocyanate represented by OCN—R¹—NCO in which R¹ is a hydrocarbyl of1-50 carbon atoms and

[0020] a second process step of producing the polysaccharide containinghydrophobic group composed of the hydrocarbon group of 12-50 carbonatoms or of the steryl group, wherein the isocyanate group-containinghydrophobic compound obtained in the first process step is reacted withone or more polysaccharides,

[0021] wherein the reaction product in the second process step ispurified using a solvent based on ketone.

[0022] (2) The process as defined in the above (1), wherein thepolysaccharide is selected from the group consisting of pullulan,amylopectin, amylose, dextran, hydroxyethyl cellulose, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan andwater-soluble cellulose.

[0023] (3) The process as defined in the above (1) or (2), wherein thesolvent based on ketone comprises one or more selected from the groupconsisting of acetone, methyl ethyl ketone, diethyl ketone anddiisopropyl ketone.

[0024] (4) The process as defined in any one of the above (1) to (3),wherein the hydrophobic group-containing polysaccharide has a grouprepresented by —XH in which X is oxygen atom or a nitrogen-containinggroup represented by NY with Y being hydrogen atom or a hydrocarbyl of1-10 carbon atoms wherein 0.1-10 —XH groups per 100 monosaccharide unitsconstituting the polysaccharide are replaced by one or more hydrophobicgroups represented by the formula (1), namely,

[0025] in which X is the same as given above, R¹ denotes a hydrocarbylhaving 1-50 carbon atoms and R² denotes a hydrocarbon group of 12-50carbon atoms or a steryl group.

[0026] (5) The process as defined in the above (4), wherein R² in theformula (1) denotes a steryl group.

[0027] (6) The process as defined in any one of the above (1) to (5),wherein the content of the hydrophobic group-containing polysaccharidein the product purified using the solvent based on ketone is as high as80% by weight or more.

[0028] (7) The process as defined in the above (6), wherein the contentof unsubstituted polysaccharide is as low as 20% by weight or less.

[0029] (8) The process as defined in the above (6) or (7), wherein theproduct has a content of the impurity product, in which both the two NCOgroups in the diisocyanate are reacted with the hydroxylgroup-containing hydrocarbon having 12-50 carbon atoms or with thesterol, as low as 0.05% by weight or less.

[0030] (9) The process as defined in any one of the above (1) to (8),wherein the product purified using a solvent based on ketone issubjected to a further purification by dispersing the product finely inwater under an ultrasonic treatment, with subsequent ultracentrifugalseparation.

[0031] (10) The process as defined in the above (9), wherein the contentof the hydrophobic group-containing polysaccharide in the purifiedproduct from the ultracentrifugal separation is as high as 98% by weightor more.

[0032] (11) The process as defined in the above (10), wherein thecontent of unsubstituted polysaccharide is as low as 2% by weight orless.

[0033] (12) The process as defined in the above (10) or (11), whereinthe content of the impurity product, in which both the two NCO groups inthe diisocyanate are reacted with the hydroxyl group-containinghydrocarbon having 12-50 carbon atoms or with the sterol, is as low as0.05% by weight or less.

[0034] (13) The process as defined in any one of the above (1) to (8),wherein the product purified using the solvent based on ketone isfurther subjected to a purification procedures comprising dissolving theproduct in an aprotic polar solvent, admixing water to the resultingsolution to cause the unsubstituted polysaccharide to be transferred tothe aqueous phase and removing the aqueous phase separated by phaseseparation.

[0035] (14) The process as defined in the above (13), wherein thefurther purification of the product purified using the solvent based onketone is performed by dissolving the product in the aprotic polarsolvent of an amount of 3-50 times the weight of the product andadmixing water to the resulting solution in an amount of at least 5times the weight of the solution.

[0036] (15) The process as defined in the above (13) or (14), whereinthe aprotic polar solvent comprises one or more selected from the groupconsisting of N,N-dimethylformamide, N,N-dimethylacetamide and dimethylsulfoxide.

[0037] (16) The process as defined in any one of the above (13) to (15),wherein the content of the hydrophobic group-containing polysaccharidein the product purified using the aprotic polar solvent is as high as98% by weight or more.

[0038] (17) The process as defined in the above (16), wherein thecontent of the unsubstituted polysaccharide is as low as 2% by weight orless.

[0039] (18) The process as defined in the above (16) or (17), whereinthe content of the impurity product, in which both the two NCO groups inthe diisocyanate are reacted with the hydroxyl group-containinghydrocarbon having 12-50 carbon atoms or with the sterol, is as low as0.02% by weight or less.

[0040] (19) A high purity product of polysaccharide containinghydrophobic group which contains at least 80% by weight of thepolysaccharide containing hydrophobic group, wherein the polysaccharideis one having a group represented by —XH in which X is oxygen atom or anitrogen-containing group represented by NY with Y being hydrogen atomor a hydrocarbyl of 1-10 carbon atoms wherein 0.1-10 —XH groups per 100monosaccharide units constituting the polysaccharide are replaced by oneor more hydrophobic groups represented by the formula (1), namely,

[0041] in which X is the same as given above, R¹ denotes a hydrocarbylhaving 1-50 carbon atoms and R² denotes a hydrocarbon group of 12-50carbon atoms or a steryl group,

[0042] the said polysaccharide containing hydrophobic group beingobtained by a process comprising

[0043] a first process step of producing an isocyanate group-containinghydrophobic compound, wherein one mole of a hydroxyl group-containinghydrocarbon having 12-50 carbon atoms or of a sterol is reacted with adiisocyanate represented by OCN—R¹—NCO in which R¹ is a hydrocarbyl of1-50 carbon atoms,

[0044] a second process step of producing the polysaccharide containinghydrophobic group composed of the hydrocarbon group of 12-50 carbonatoms or of the steryl group, wherein the isocyanate group-containinghydrophobic compound obtained in the first process step is reacted withone or more polysaccharide, and

[0045] purifying the reaction product from the second process step usinga solvent based on ketone.

[0046] (20) The high purity product of polysaccharide containinghydrophobic group as defined in the above (19), wherein thepolysaccharide is selected from the group consisting of pullulan,amylopectin, amylose, dextran, hydroxyethyl cellulose, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan andwater-soluble cellulose.

[0047] (21) The high purity product of polysaccharide containinghydrophobic group as defined in the above (19) or (20), wherein R² inthe formula (1) is steryl.

[0048] (22) The high purity product of polysaccharide containinghydrophobic group as defined in any one of the above (19) to (21),wherein the content of unsubstituted polysaccharide is as low as 20% byweight or less.

[0049] (23) The high purity product of polysaccharide containinghydrophobic group as defined in any one of the above (19) to (22),wherein the content of the impurity product, in which both the two NCOgroups in the diisocyanate are reacted with the hydroxylgroup-containing hydrocarbon having 12-50 carbon atoms or with thesterol, is as low as 0.05% by weight or less.

[0050] (24) The high purity product of polysaccharide containinghydrophobic group as defined in any one of the above (19) to (23),wherein the product purified using the solvent based on ketone issubjected to a further purification by dispersing the product finly inwater under an ultrasonic treatment, with subsequent ultracentrifugalseparation.

[0051] (25) The high purity product of polysaccharide containinghydrophobic group as defined in any one of the above (19) to (23),obtained by subjecting the product purified using the solvent based onketone to a further purification procedures comprising dissolving theproduct in an aprotic polar solvent, admixing water to the resultingsolution to cause the unsubstituted polysaccharide to be transferred tothe aqueous phase and removing the aqueous phase separated by phaseseparation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 shows ¹H-NMR spectrum of the pullulan-cholesterolderivative (CHP) obtained in Example 1-2.

[0053]FIG. 2(a) shows the result of analysis by a size exclusionchromatography (SEC) of a sample of the pullulan-cholesterol derivative(CHP) obtained in Example 1-2 before the ultrasonic treatment and FIG.2(b) shows the result of SEC analysis of a sample collected after 30minutes from the ultrasonic treatment. FIG. 2(c) shows the result of SECanalysis of the supernatant of an ultracentrifugation and FIG. 2(d)shows the result of SEC analysis of the solution resulting from anultrasonic treatment of the aqueous phase of the bottom layer of theultracentrifugation which has been re-swollen with water. In theseFigures, the ordinate represents the strength (dimensionless) of thedifferential refractometer reading (the same applies in the following).

[0054]FIG. 3 shows ¹H-NMR spectrum of the mannan-cholesterol derivative(CHM) obtained in Example 2-1.

[0055]FIG. 4 shows ¹H-NMR spectrum of the mannan-cholesterol derivative(CHM) obtained in Example 2-2.

[0056]FIG. 5 shows the result of analysis by a size exclusionchromatography (SEC) of a sample of the pullulan-cholesterol derivative(CHP) obtained in Example 1-2 which is purified with a non-polar solventfollowed by an ultrasonic treatment.

THE BEST MODE FOR EMBODYING THE INVENTION

[0057] In the context of this specification, it is meant by “highpurity” that the contents of the dimer resulting from the reaction ofthe hydrophobic group, such as the hydrocarbon group or the sterylgroup, with the diisocyanate compound and the unsubstitutedpolysaccharide are scarce.

[0058] The hydroxyl group-containing hydrocarbon having 12-50 carbonatoms to be incorporated according to the present invention is used asthe raw material for the introduction of hydrophobic group. As thehydroxyl group-containing hydrocarbon group having 12-50 carbon atoms tobe incorporated according to the present invention, there may beexemplified those originated from alcohols, such as lauryl alcohol,myristyl alcohol, cetyl alcohol, stearyl alcohol, arachinyl alcohol,docosanol, pentacosanol, hexacosanol and octacosanol. Among them,preference is given to alcohols having 12-35 carbon atoms, especiallyhaving 12-20 carbon atoms, due to their easy availability. The hydroxylgroup-containing hydrocarbon group having 12-50 carbon atoms may beemployd either alone or in a combination of two or more of them. When ahydroxyl group-containing hydrocarbon group having less than 12 carbonatoms is employed as the raw material for the introduction ofhydrophobic group, the coagulating effect by hydrophobicity becomesunfavorably difficult to be revealed sufficiently. In contrast, if thenumber of carbon atoms exceeds over 50, such a product is difficultlyavailable and unfavorable.

[0059] The sterol to be incorporated according to the present inventionis used as the raw material for the introduction of the hydrophobicgroup. As the sterol to be employed according to the present invention,there may be enumerated, for example, cholesterol, stigmasterol,β-sitosterol, lanosterol and ergosterol. Among them, preference is givento cholesterol for its availability and so on. Sterols may be employedeither alone or in a combination of two or more of them. It ispermissible to use the sterol together with the hydroxylgroup-containing hydrocarbon group having 12-50 carbon atoms.

[0060] The diisocyanate compound to be incorporated according to thepresent invention is that represented by the formula OCN—R¹—NCO (inwhich R¹ denotes a hydrocarbyl having 1-50 carbon atoms). Such a numberof carbon atoms as exceeding over 50 is undesirable, since such adiisocyanate is difficultly obtainable. Concrete examples of thediisocyanate compound include ethylene diisocyanate, namely, with R¹ ofethylene, butylene diisocyanate, with R¹ of butylene, hexamethylenediisocyanate, with R¹ of hexamethylene, and diphenyl-methanediisocyanate, with R¹ of diphenylmethane.

[0061] For the polysaccharide to be incorporated according to thepresent invention, those of natural occurrence and semisynthetic originmay be employed. Concretely, there may be exemplified one or moreselected from the group consisting of pullulan, amylopectin, amylose,dextran, hydroxyethyl cellulose, hydroxyethyl dextran, mannan, levan,inulin, chitin, chitosan, xyloglucan and water-soluble cellulose. Amongthem, pullulan, mannan, xyloglucan, amylopectin, dextran andhydroxyethyl cellulose are preferred. Also, those nitrogen-containingpolysaccharides, such as chitin, partial deacetylated chitin andchitosan, are favorable. The polysaccharides may be employed eitheralone or in a combination of two or more of them.

[0062] The hydrophobic group-containing polysaccharides produced by theprocess according to the present invention are those, in which 0.1 to10, preferably 0.1 to 6 —XH groups, per 100 monosaccharide unitsconstituting the polysaccharide having one or more groups represented by—XH (in which X is oxygen atom or a nitrogen-containing grouprepresented by NY with Y being hydrogen atom or a hydrocarbyl of 1-10carbon atoms) are replaced by the hydrophobic group represented by theformula (1) given above.

[0063] In the formula (1), R¹ denotes a group originated from thediisocyanate compound mentioned above. R² represents a hydroxylgroup-containing hydrocarbon group having 12-50 carbon atoms and/or agroup originated from sterol. Concrete examples of the group representedby R² include laulyl, myristyl, cetyl, stearyl, cholesteryl,stigmasteryl, β-sito-steryl, lanosteryl and ergosteryl. More preferably,myristyl, stearyl and cholesteryl are enumerated.

[0064] In the production process according to the present invention, thehydroxyl group in the CH₂OH group and the hydroxyl group bound directlyto the monosaccharide are both subject to the replacement with thehydrophobic group represented by the formula (1), when a polysaccharidein which the constituent monosaccharides have CH₂OH group bound thereto,such as pullulan, mannan or so on, is employed, wherein the proportionof the group subjected to such replacement is far greater for thehydroxyl group in the CH₂OH group than the hydroxyl group bound directlyto the monosaccharide.

[0065] In case a polysaccharide having CH₂OH groups and NH₂ groups boundthereto, such as chitosan or the like, is employed, the OH group inCH₂OH, NH₂ group and the OH group bound directly to the monosaccharideare all subject to the replacement with the hydrophobic grouprepresented by the formula (1), wherein the proportion of the groupsubjected to such replacement is far greater for the OH group in CH₂OHand for the NH₂ group than for the OH group bound directly to themonosaccharide.

[0066] The production process according to the present inventioncomprises either the process steps 1 to 3 or the process steps 1 to 4 asgiven below. While the description of the production process in thefollowing is directed to the case of using pullulan as thepolysaccharide and a steryl group as the hydrophobic group, theproduction can be realized in a similar way also with other ones. Thereactions included in the production process according to the presentinvention are given by the reaction schemes (I) and (II), wherein thereaction scheme (I) corresponds to the process step 1 and the reactionscheme (II) to the step 2.

[0067] Process step 1

[0068] As shown in the reaction scheme (I), the steryl isocyanate to beemployed according to the present invention is a compound represented bythe formula (4) having the steryl group at one end of an alkane and anisocyanate group at the other end thereof and can be obtained by thereaction of a diisocyanate compound represented by the formula (2) and asterol expressed by the formula (3). On producing the compoundrepresented by the formula (4), one isocyanato group of the diisocyanatecompound represented by the formula (2) is reacted with the hydroxylgroup of the sterol expressed by the formula (3) to produce the sterylisocyanate of the formula (4) in which the sterol is bound via anurethane bond at one end and the isocyanate group at the other endremains unreacted as such. In this reaction, sterol dimer represented bythe formula (5) is by-produced usually at a proportion of about 10% byweight.

[0069] Process step 2

[0070] As shown in the reaction scheme (II), the steryl isocyanaterepresented by the formula (4) obtained in the process step 1 givenabove is reacted with a polysaccharide (pullulan) represented by theformula (6) to produce the polysaccharide-sterol derivative (hydrophobicgroup-containing polysaccharide) represented by the formula (7). Thisreaction is addition of the hydroxyl group of the polysacchariderepresented by the formula (6) and the isocyanato group of the sterylisocyanate represented by the formula (4) in an organic solvent in thepresence of a basic catalyst.

[0071] Process step 3

[0072] The reaction product obtained by the above process step 2 ispurified by reprecipitation thereof using a solvent based on ketone (inthe following, this purification is called “ketone-purification”). Bythe ketone-purification, principally the sterol dimer by-produced in theprocess step 1 is removed, whereby a high purity product ofpolysaccharide-sterol derivative (the hydrophobic group-containingpolysaccharide) can be obtained. In the specification of the presentinvention, the product resulting from the ketone-purification isexpressed as “ketone-purified product”. The ketone-purified product canalso be subjected to a further purification by dialysis, in order toremove the reaction solvent.

[0073] Process step 4

[0074] 1) The ketone-purified product ontained in the above process step3 (including the purified product resulting from the dialysis) issubjected to a further purification by an ultracentrifugation. By thepurification by ultracentrifugation, predominantly the unsubstitutedpolysaccharide (unreacted polysaccharide) is removed, whereby a morehighly purified product of the hydrophobic group-containingpolysaccharide can be obtained.

[0075] 2) In the production process according to the present invention,the above purification procedure 1) by means of an ultracentrifugationcan be replaced by a purification procedure using an aprotic polarsolvent. The purification technique using the aprotic polar solventconsists in the procedures comprising dissolving the ketone-purifiedproduct (inclusive of the product purified by the dialysis) obtained bythe above process step 3 in an aprotic polar solvent added thereto,introducing water to the resulting solution with sufficient agitationby, such as stirrer, and removing the aqueous layer formed by the phaseseparation. By the purification using the aprotic polar solvent, theunsubstituted polysaccharide (unreacted polysaccharide) is removedpredominantly, whereby a still more highly purified hydrophobicgroup-containing polysaccharide can be obtained. This procedure can beeffected in any repeats and, by a few repeats, the purity of thehydrophobic group-containing polysaccharide is further improved. It isalso possible to obtain the hydrophobic group-containing polysaccharidein a solid pulverous form by removing the aprotic polar solvent.

[0076] Below, the process steps according to the present invention willfurther be described in more detail.

[0077] The step of producing the compound represented by the fromula (4)in the process step 1 consists in the reaction of the diisocyanatecompound represented by the formula (2) with the sterol represented bythe formula (3) in an organic solvent in the presence of a basiccatalyst. The amount of the diisocyanate compound to be used mayfavorably be 1-30 molar equivalents, preferably 10-20 molar equivalents,per one mole of the sterol. As the basic catalyst, use of amines isfavorable, whereby the reaction proceeds efficiently.

[0078] As the organic solvent to be employed in the reaction, there maybe enumerated, for example, solvents based on ether, aprotic polarsolvents, solvents based on halogen compound and solvents based onaliphatic hydrocarbon and based on aromatic hydrocarbon. As the solventbased on ether, there may be exemplified apliphatic ethers, such asethyl ether and so on, and heterocyclic ethers, such as tetrahydrofuranand the like. As the aprotic polar solvent, there may be enumerated, forexample, acetone, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).As the solvent based on halogen compound, there may be exemplifiedmethylene chloride and chloroform. As the aliphatic hydrocarbon solvent,pentane, hexane and so on are exemplified. As the aromatic hydrocarbonsolvent, benzene, toluene and so on are enumerated. Among them, aromatichydrocarbons are preferred.

[0079] As the amine to be employed in the reaction of the reactionscheme (I), triethylamine, pyridine and so on may be exemplified. Theamount of the amine to be used may range from 1 to 20 molar equivalents,preferably from 1 to 3 molar equivalents, per one mole of the sterol.The temperature and the duration of the reaction may differ inaccordance with, for example, the diisocyanate compound and the solventused, and may be settled with respect to the condition of progress ofthe reaction, while the reaction temperature may range preferably fromroom temperature to 100° C. and the reaction duration may preferably bein the range of 3-24 hours.

[0080] For the reaction, it is preferable to employ a dried solvent anda dried basic catalyst with preference to the condition under an inertgas atmosphere. As the inert gas, for example, nitrogen, argon and thelike are enumerated.

[0081] The step of producing the compound represented by the formula (7)in the process step 2 consists in the reaction of the polysacchariderepresented by the formula (6) with the steryl isocyanate compoundrepresented by the formula (4) produced in the above process step 1 inan organic solvent in the presence of a basic catalyst. While theproportion of the charge amount of the polysaccharide and that of thesteryl isocyanate is determined here by the contemplated amount of thesteryl group to be introduced into the polysaccharide, preference isgiven to a ratio in the range from 0.1 to 10 molar equivalent withrespect to 100 monosaccharide units in the polysaccharide.

[0082] As organic solvent to be used for the reaction, there may beenumerated, forexample, solvents based on ether, aprotic polar solvents,solvents based on halogen compound and solvents based on aliphatichydrocarbon and based on aromatic hydrocarbon. As the solvent based onether, there may be exemplified apliphatic ethers, such as ethyl etherand so on, and heterocyclic ethers, such astetrahydrofuran and the like.As the aprotic polar solvent, there may be enumerated, for example,acetone, dimethylformamide (DMF) and dimethyl sulfoxide(DMSO). As thesolvent based on halogen compound, theremay be exemplified methylenechloride and chloroform. As the aliphatic hydrocarbon solvent, pentane,hexane and so on are exemplified. As the aromatic hydrocarbon solvent,benzene, toluene and so on are enumerated. Among them, aprotic polarsolvents are preferred.

[0083] As the basic catalyst to be employed in the reaction of thereaction scheme (II), amines are preferred and, for example,triethylamine, pyridine and so on may be enumerated. The amount of theamine to be used may range from 1 to 10 molar equivalents, preferablyfrom 1 to 3 molar equivalents, per one mole of the polysaccharide. Thetemperature and the duration of the reaction may differ in accordancewith, for example, the polysaccharide and the solvent used, and may besettled with respect to the condition of progress of the reaction, whilethe reaction temperature may range preferably from room temperature to100° C. and the reaction duration may preferably be in the range from 30minutes to 24 hours.

[0084] For the reaction, it is preferable to employ a dried solvent anda dried basic catalyst with preference to the condition under an inertgas atmosphere. As the inert gas, for example, nitrogen, argon and thelike are enumerated.

[0085] As the solvent based on ketone to be employed in theketone-purification in the above process step 3, there may be enumeratedat least one selected from the group consisting of acetone, methyl ethylketone, diethyl ketone, diisopropyl ketone and the like. The amount ofthe solvent based on ketone to be used may be 4-50 times, preferably8-20 times, the weight of the reaction solution obtained in the aboveprocess step 2. When the reaction product obtained in the above processstep 2 is added to a solvent based on ketone, the polysaccharide-sterolderivative (the hydrophobic group-containing polysaccharide) willprecipitate out and the sterol dimer by-produced in the process step 1is dissolved in the solvent based on ketone, so that a high purityproduct of the polysaccharide-sterol derivative can be obtained byseparating and collecting the precipitate. The precipitate can be driedby a technique, such as freeze-drying, vacuum drying or the like. By theketone-purification, a higher rate of removal of the sterol dimer can beachieved as compared with the conventional technique, such as dialysis,reprecipitation using ethanol, column chromatography or so on, whereby ahigh purity product of the hydrophobic group-containing polysaccharidecan be obtained in an easy manner.

[0086] The content of the hydrophobic group-containing polysaccharide inthe ketone-purified product is as high as 80% by weight or more,preferably as high as 90% by weight or more. The content of theunsubstituted polysaccharide is as low as 20% by weight or less,preferably as low as 10% by weight or less. The content of the impurityresulting from the reaction of both the two NCO groups in thediisocyanate compound with the hydrophobic group amounts to as low as0.05% by weight or less, preferably as low as 0.01% by weight or less.

[0087] In the purification technique by means of an ultracentrifugationin the above process step 4-1), the ketone-putified product from theprocess step 3 is subjected to an ultracentrifugation after anultrasonic treatment of the product with addition of water thereto.Here, distilled water, deionized water and the like may be used. Theamount of water to be added may be 5-100 times, preferably 30-60 timesthe weight of the ketone-putified product. The ultracentrifugation maypreferably be carried out at an acceleration of 10,000-200,000 G,preferably 30,000-100,000 G for a period of time of 1-24 hours,preferably 3-15 hours. By an ultracentrifugation, a phase separationoccurs wherein the polysaccharide-sterol derivative having greatermolecular weight gathers in the lower layer and the unsubstitutedpolysaccharide having lower molecular weight moves to the upper layer,whereby the high purity product of the polysacchareide-sterol derivativecan be obtained by collecting the lower layer.

[0088] The content of the hydrophobic group-containing polysaccharide inthe purified product from the ultracentrifugation is as high as 98% byweight or more, preferably as high as 99.9% by weight or more. Thecontent of the unsubstituted polysaccharide is as low as 2% by weight orless, preferably as low as 0.1% by weight or less. The content of theinpurity resulting from the reaction of both two NCO-groups in thediisocyanate compounds with the hydrophobic group is as low as 0.05% byweight or less, preferably as low as 0.01% by weight or less. By thepurification by ultracentrifugation, even a high purity product of 99.9%by weight or higher can easily be obtained.

[0089] As the aprotic polar solvent which can be employed in thepurification of the process step 4-2) using the aprotic polar solvent,there may be enumerated one or more selected from the group consistingof, for example, N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMAc), 1,3-dimethyl-2-imidazolidinon (DMI), dimethyl sufoxide (DMSO)and so on. The amount of the aprotic polar solvent to be employed may be3-50 times, preferably 5-15 times, the weight of the ketone-purifiedproduct obtained in the process step 3. If the amount is less than 3times the weight, it is short for dissolving the ketone-purifiedproduct. When the amount exceeds 50 times the weight, no phaseseparation into two phases may occur upon the addition of water,remaining in a miscible state. The temperature for dissolving theketone-purified product in the aprotic polar solvent may be in the rangeof 0-150° C., preferably from room temperature to 100° C. Use of solventother than the aprotic polar solvent is unfavorable, since thehydrophobic group-containing polysaccharide and the unsubstitutedpolysaccharide may not be dissolved therein.

[0090] As the water to be used in the above process step 4-2), distilledwater, deionized water, pure water and so on may be enumerated. Here,the amount of water to be used may be at least 5 times, preferably10-100 times the weight of the solution obtained by dissolving theketone-purified product in the aprotic polar solvent. If the amount isshort of 5 times the weight, no phase separation may occur and themixture remains in a miscible state. When, in contrast, the amount ofwater exceeds 100 times the weight, no marked increase in the efficiencyof removal of the unsubstituted polysaccharide can be expected. It ispreferable to add a predetermined amount of water to the solution all atonce with subsequent mechanical agitation by, for example, stirrer. Amanner of addition with simultaneous mechanical agitation is notpreferred, since a phase separation into two phases is therebycounteracted by the formation of miscible mixture. It is permissible toperform the phase separation procedure by means of standing the mixturestill or by means of a forced phase separation by, such ascentrifugation.

[0091] When water is added to the solution of the ketone-purifiedproduct in the aprotic polar solvent and agitating the resulting mixtureto cause occurrence of phase separation into two layers of an aqueouslayer and an aprotic polar solvent layer, the unsubstitutedpolysaccharide will move into the aqueous layer and the objectivehydrophobic group-containing polysaccharide remains dissolved in theaprotic polar solvent layer, so that the unsubstituted polysaccharidecan be removed by separating off the aqueous layer. By removing theaprotic polar solvent from the solvent layer remained after the removalof the aqueous layer, a high purity product of the hydrophobicgroup-containing polysaccharide can be obtained. For removing theaprotic polar solvent, there may be employed techniques, such aschromatography, freeze-drying and reprecipitation. In particular, inreprecipitation, use of a solvent based on ketone or based on alcohol asthe anti-solvent is preferred, for example, acetone, methyl ethylketone, methanol, ethanol and so on. The amount of the anti-solvent maybe 4-50 times, preferably 8-20 times, the weight of the solution to besubjected to the reprecipitation. The resulting precipitate can be driedby techniques, such as freeze-drying and vacuum drying.

[0092] The purification using an aprotic polar solvent can be performedmore conveniently in a more efficient manner as compared with thepurification technique by means of an ultracentrifugation of the aboveprocess step 4-1). By this, a more larger scale production of highpurity product of the hydrophobic group-containing polysaccharideexpected for the use in living body, such as for a medicamment, becomespossible.

[0093] The content of the hydrophobic group-containing polysaccharide inthe purified product resulting from the technique using an aprotic polarsolvent may be as high as 98% by weight or higher, preferably as high as99.9% by weight or higher. The content of the unsubstitutedpolysccharide is as low as 2% by weight or less, preferbly as low as0.1% by weight or less. The content of the impurity resulting from theside reaction in which the two NCO groups in the diisocyanate compoundhave been reacted with the hydrophobic group may be at most 0.02% byweight, preferably at most 0.01% by weight. By the purification using anaprotic polar solvent, a high purity product of 99.9% by weight orhigher can easely be obtained.

[0094] By the process according to the present invention as describedabove, it is made possible to remove the by-poduced sterol dimer, whichremains in the final product and the complete removal of which hasheretofore been quite difficult by prior techniques, in a convenient andefficient manner, wherby production of a high purity product ofhydrophobic group-containing polysaccharide can be realized.

[0095] The high purity polysaccharide containing hydrophobic groupaccording to the present invention is that obtained by the processaccording to the present invention described above, which has thehydrophobic group represented by the foumula (1) given above and has apurity as high as 80% or more, preferably as high as 90% or more. Thehigh purity polysaccharide according to the present invention can form afinely dispersed colloid solution by its cohesive nature due to thehydrophobic group represented by the formula (1) and reveals thus anability for building up polymer micelles of a core/shell type.

[0096] The high purity polysaccharide containing hydrophobic groupaccording to the present invention can be utilized as coating materialfor coating drug carrier having encapsulated therein a drug. It can beused as the coating material for coating drug carrier, such as liposomemicrocapsule, microsphere, O/W emulsion and erythrocyte ghost. The highpurity polysaccharide containing hydrophobic group can be used for suchmedical materials safely, since it has scarce contents of by-productsand of the unsubstituted polysaccharide and is highly pure.

[0097] As described above, the process for producing the high puritypolysaccharide containing hydrophobic group according to the presentinvention permits to produce a high purity product of polysaccharidecontaining hydrophobic group having scarce content of impurities, suchas unsubstituted polysaccharide and sterol dimer, in an easy andefficient manner, since it includes a step of purification using asolvent based on ketone. A more highly pure product of polysaccharidecontaining hydrophobic group can be produced by incorporating, incombination, a purification by an ultracentrifugation or a purificationwith an aprotic polar solvent.

[0098] The high purity polysaccharide according to the present inventionhas a high purity due to its production process as described above, sothat it can be used as a medical material safely.

[0099] Below, the present invention will further be described moreconcretely by way of examples.

EXAMPLE 1-1 Synthesis of N-(6-isocyanatohexyl)cholesteryl Carbamate

[0100] In an eggplant type flask of 1 liter capacity, there are charged25 g (0.065 mole) of cholesterol and thereto were added 300 ml oftoluene to dissolve it, whereto 17 ml (0.12 mole) of triethylamine wereadded. To this mixture, there were added 161 g (0.96 mole, 14.8 eq.) ofhexamethylene diisocyanate dissolved in 300 ml of toluene and theresulting mixture was subjected to reaction at 80° C. under a nitrogenatmosphere for about 6 hours. After termination of the reaction, tolueneand the excess of hexamethylene diisocyanate were removed under areduced pressure. By standing the resulting yellowish oily residue stillat room temperature overnight, pale yellow crystals were formed. Thecrystals were taken out and introduced into about one liter of hexaneand the mixture was shaken vigorously, whereupon the supernatant wasremoved by decantation. This washing procedure was repeated four times,whereupon the product was dried under a reduced pressure for three hoursat room temperature, whereby a white solid product (crystals) wasobtained. Yield: 18.25 grams (50.9%)

[0101] The results of analysis of this product by ¹H-NMR and IR were asgiven below.

[0102]¹H-NMR: δ ppm, in CDCl₃, TMS 0.68-2.35 (m, 43H) 1.34-1.55 (m, 8H)3.14-3.18 (m, 2H) 3.27-3.32 (t, J=6.6 Hz, 2H) 4.4-4.6 (m, 2H) 5.38 (m,1H) IR (KBr, cm⁻¹):3260, 2320, 1680, 1130

[0103] From these data, it was confirmed that the product obtained isN-(6-isocyanatohexyl)cholesteryl carbamate represented by the followingformula (4a):

[0104] On the other hand, the white crystls obtained were examined by athin layer chromatography by developing it using Preparative TLC(supplied from Merck AG, Silika gel 60 F₂₅₄, developer: hexane/ethylacetate=2/1), whereby existence of the by-produced cholesterol dimer(Rf=0.65) represented by the following formula (5a) was confirmed. Theband for the cholesterol dimer on the TLC was subjected to extractionwith acetone and the extract was analyzed, whereby it was confirmed thatthe cholesterol dimer was contained in the white crystalline product inan amount of 8% by weight.

EXAMPLE 1-2 Synthesis of Pullulan-cholesterol Derivative (CHP)

[0105] In an eggplant type flask of 1 liter capacity, there are charged40 g (248 mmol as anhydrous glucose unit) of pullulan (average molecularweight: 108,000) and 420 ml of dimethyl sulfoxide (sometimes abbreviatedas DMSO) were added thereto and the mixture was agitated at 80° C. undera nitrogen atmosphere to dissolve it. To this mixture, a solution of1.78 g (3.21 mmol) of N-(6-isocyanotohexyl)cholesteryl carbamatesynthesized in EXAMPLE 1-1 dissolved in 31.6 g (0.40 mol) of pyridinewas added and the mixture was subjected to reaction at 90° C. for 3hours.

[0106] After termiantion of the reaction, dimethyl sulfoxide wasdistilled off and the resulting oily residue was dropped into 6 litersof acetone to form a precipitate to purify the product. After removal ofthe supernatant, 4 liters of acetone were added to the resultingprecipitate and the mixture was stood still overnight at roomtemperature. The precipitate was collected by filtration and was driedunder a reduced pressure. The so-obtained solids were dissolved indimethyl sulfoxide and the solution was charged in a dialysis bag(Spectra/Por3, a product of the frim Spectropor, an exclusion molecularweight of 3,500) and was subjected to a dialysis against distilled waterfor one week. 1.5 liters of the resulting polymer solution were treatedby freeze-drying in an ordinary manner, whereby a white solid matter (inthe following, denoted occasionally as “acetone-purified product”) wasobtained. Yield: 31.7 g (76.2%)

[0107] The results of analyses of this acetone-purified product by¹H-NMR and IR were as given below.

[0108]¹H-NMR: δ ppm, DMSO-d₆/D₂O=20/1, vol. 0.68-2.40 2.60-4.604.70-5.30 IR (KBr, cm⁻¹): 1680, 1180-900

[0109] From these data, it was confirmed that the product obtained is apullulan/chlosterol derivative (abbreviated hereinafter sometimed asCHP) represented by the following formula (7a):

[0110] On the other hand, the above product purified with acetone wereanalyzed by a thin layer chromatography using Preparative TLC {suppliedfrom Merck AG, Silika gel 60 F₂₅₄, developer: hexane/ethyl acetate(2/1)}, whereby existence of the by-produced cholesterol dimer (Rf=0.65)represented by the formula (5a) was not recognized. Further, theacetone-purified product was analysed by ¹H-NMR and the content of thecholesterol dimer was calculated from the protone ratio. From theresult, existence of the cholesterol dimer was not recognized. Thereforethe content of the cholesterol dimer represented by the formula (5a) inthe acetone-purified product is 0% by weight.

[0111] The acetone layer used for the purification was collected and theamount of cholesterol dimer contained therein was estimated. It wasfound that the amount of cholesterol dimer contained in the acetonelayer was 0.140 gram. Since the amount of cholesterol dimer contained inthe raw material of 1.78 grams (3.21 mmol) ofN-(6-isocyanatohexyl)cholesteryl carbamate is calculated to be 0.142gram (It had been confirmed that the content of cholesterol dimer in theproduct of EXAMPLE 1-1 was 8% by weight), the rate of removal of thecholesterol dimer is calculated from these data. Result is recited inTable 1.

[0112] The ¹H-NMR spectrogram of the resulting target compound, i.e.,pullulan-cholesterol derivative represented by the formula (7a), isgiven in FIG. 1. From the integration value of the peak area of this¹H-NMR spectrogram, the proportion of introduction of the cholesterolgroups into pullulan of the pullulan-cholesterol derivative iscalculated by the following calculation equation (A):

(100+2x)/51x=b/a  (A)

[0113] in which the symbols denote:

[0114] a: the peak area of the cholesterol group (δ=0.68-2.40)

[0115] b: the peak area of pullulan (δ=4.70-5.30)

[0116] x: proportion of substitution with cholesterol group per 100monosaccharide units.

[0117] From the calculation, it is found that the proportion ofsubstitution with cholesterol group in the pullulan-cholesterolderivative represented by the above formula (7a) is 1.1 groups per 100monosaccharide units.

Comparative Example 1 Test of Reprecipitation with Ethanol

[0118] A pullulan-cholesterol derivative was synthesized by the sameprocedures as in EXAMPLE 1-2. After termination of the reaction, thepurification was performed by reprecipitation with ethanol, whereby apullulan-cholesterol derivative was obtained.

[0119] The analysis of the product purified with ethanol was performedusing Preparative TLC in the same manner as in EXAMPLE 1-2. From theresult, existence of the cholesterol dimer (Rf=0.65) represented by theformula (5a) was confirmed. The content of the cholesterol dimer in theproduct purified with ethanol was calculated from ¹H-NMR analysis as inEXAMPLE 1-2. The result showed that the cholesterol dimer is containedin the product in an amount of 0.4% by weight.

[0120] The ethanol layer used for the purification was collected and theamount of the cholesterol dimer contained therein was estimated, whichshowed that the content was 0.016 gram. From this, the rate of removalof the cholesterol dimer was calculated in the same manner as in EXAMPLE1-2. The result is recited in Table 1. TABLE 1 EXAMPLE COMP. 1-2 EXAMPLE1 Solvent for reprecipitation Acetone Ethanol Colesterol dimer content(wt. %) 0 0.4 Cholesterol dimer removal wt. % 98.6 11.3

[0121] From Table 1, it is confirmed that the cholesterol dimer can beremoved almost completely by purifying the product by reprecipitationusing acetone as the reprecipitation solvent.

EXAMPLE 1-3 Purification of Pullulan-cholesterol Derivative (CHP)

[0122] To 40 mg of the pullulan-cholesterol derivative synthesized inEXAMPLE 1-2, 20 ml of pure water were added and the mixture wassubjected to an ultrasonic wave irradiation by a sonicator of aprobe-type (TOMY, a unit supplied by the firm URP, with a probe outerdiameter of 5 mm) for 30 minutes at 40 W. During the irradiation, thetemperature of the aqueous mixture was maintained at 4° C. by coolingthe vessel from outside with ice water.

[0123] Samples of each 10 ml of the ultrasonic wave-irradiated aqueousmixture were collected in centrifuge tubes and were subjected to acentrifugation at 55,000 G for 10 hours at 25° C. A phase separation wasbrought about, wherein the unsubstituted pullulan (unreacted pullulan)was gathered in the supernatant and the pullulan-cholesterol derivative(CHP) was separated in the lower layer.

[0124] The samples collected before and after the ultrasonic waveirradiation, respectively, were analysed by SEC (size exclusionchromatography). The conditions in the SEC were as given below. Theresults are shown in FIGS. 2(a) and 2(b), respectively. Apparatus used:TOSOH HPSEC SYSTEM (trademark, of Tosoh K. K. ) Column: TSK-gelG4000SWXL (trademark, of Tosoh K. K. ) Eluent: 0.02% NaN₃ in deionizedwater Flow rate: 0.5 ml/min. Temperature: 35° C. Detector: RI(differential refractometer)

[0125] Calculated from the peak area of FIG. 2(b), it is found thatpullulan of which molecular weight is lower (unreacted pullulan) iscontained in the product purified with acetone in an amount of about 5%by weight. The supernatant of ultracentrifugation was subjected to anSEC analysis. The result is shown in FIG. 2(c). The gelled mass(precipitate) in the lower layer of the centrifugation was caused toswell again with water and was then subjected to an ultrasonic wavetreatment in the same manner as above, whereupon the so-ultrasonicatedsolution was examined by SEC analysis. The result is shown in FIG. 2(d).From these results, it is confirmed that, in the supernatant of theultracentrifugation, almost 100% by weight of the lower molecular weightpullulan (unreacted pullulan) as impurity have been removed and that theprecipitate has no content of the lower molecular weight pullulan.

[0126] From the above results, it was confirmed that thepullulan-cholesterol derivetive (CHP) represented by the formula (7a)was obtained at a high purity. The results are summarized in Table 2.TABLE 2 Content of CHP in acetone-purified 95 product (wt. %) Content ofCHP in ultracentrifug.- 100 purified product (wt. %) Content ofunsubstituted pullulan 0 (wt. %) Content of cholesterol dimer 0 (wt. %)

EXAMPLE 2-1 Synthesis 1 of Mannan-cholesterol Derivative (CHM)

[0127] By following the same procedures for the reaction as in EXAMPELE1-2, a commercial product of mannan (a product of the firm Sigma) andN-(6-iso-cyanatohexyl)cholesteryl carbamate were brought into rection.The charged amount of each starting material was as given below:

[0128] 1) Mannan (Mw=85,000): 26.2 g (162 mmol as anhydrous mannoseunit)

[0129] 2) N-(6-isocyanatohexyl)cholesteryl carbamate: 1.08 g (1.95 mmol)

[0130] 3) Pyridine: 19.2 g (243 mmol)

[0131] 4) Dimethyl sulfoxide: 320 ml

[0132] After termination of the reaction, purification was performed byreprecipitation with acetone solvent. Then, the product was subjected toa dialysis, followed by a freeze-drying, whereby 21.5 grams(yield=79.5%) of a white solid matter was obtained.

[0133] The results of analyses of the above acetone-purified product by¹H-NMR and IR are given below:

[0134]¹H-NMR: δ ppm, DMSO-d6/D₂O=20/1, vol. 0.68-2.40 2.60-4.604.60-5.40 IR (KBr, cm⁻¹): 1680, 1180-900

[0135] From these data, it was confirmed that the product obtained is amannan/chlosterol derivative (CHM) represented by the following formula(7b):

[0136] On the other hand, the above product purified with acetone wasanalyzed using Preparative TLC in the same manner as in EXAMPLE 1-2,whereby existence of the cholesterol dimer (Rf=0.65) represented by theformula (5a) was not recognized. Further, the acetone-purified productwas analyzed by ¹H-NMR and the content of the cholesterol dimer wascalculated from the proton ratio. From the result, existence of thecholesterol dimer was not recognized. Therefore the content of thecholesterol dimer represented by the formula (5a) in theacetone-purified product is 0% by weight.

[0137]¹H-NMR spectrogram of the compound represented by the aboveformula (7b) obtained in the manner described above is shown in FIG. 3.The proportion of introduction of the cholesteryl group into mannan inthe mannan-cholesterol derivative is calculated in the same way as inEXAMPLE 1-2. From the result, it is found that the proportion ofsubstitution with cholesteryl group is 1.1 groups per 100 monosaccharideunits.

[0138] Then, the acetone-purified product obtained as above was furtherpurified by an ultracentrifugation in the same manner as in EXAMPLE 1-3to remove the lower molecular weight mannan (unsubstituted mannan). Theresults are given in Table 3. TABLE 3 Content of CHM in acetone-purified90 product (wt. %) Content of CHM in ultracentrifug.- 100 purifiedproduct (wt. %) Content of unsubstituted mannan 0 (wt. %) Content ofcholesterol dimer (wt. %) 0

EXAMPLE 2-2 Synthesis 2 of Mannan-cholesterol Derivative (CHM)

[0139] By following the same procedures for the reaction as in EXAMPELE1-2, a commercial product of mannan (a product of the firm Sigma) andN-(6-iso-cyanatohexyl)cholesteryl carbamate were brought into rection.The charged amount of each starting material was as given below:

[0140] 1) Mannan (Mw=85,000): 5 g (31 mmol as anhydrous mannose unit)

[0141] 2) N-(6-isocyanatohexyl)cholesteryl carbamate:

[0142] 138 mg (0.25 mmol)

[0143] 3) Pyridine: 3.7 g (47 mmol)

[0144] 4) Dimethyl sulfoxide: 75 ml

[0145] After termination of the reaction, purification was performed byreprecipitation with acetone solvent. Then, the product was subjected toa dialysis, followed by a freeze-drying, whereby 4.05 grams (yield=78.8%of a white solid matter was obtained.

[0146] The results of analyses of the above acetone-purified product by¹H-NMR and IR are given below:

[0147]¹H-NMR: δ ppm, DMSO-d₆/D₂O=20/1, vol. 0.68-2.40 2.60-4.604.60-5.40 IR (KBr, cm⁻¹): 1680, 1180-900

[0148] From these data, it was confirmed that the product obtained is amannan/chlosterol derivative (CHM) represented by the above formula(7b):

[0149] On the other hand, the above product purified with acetone wasanalyzed using Preparative TLC in the same manner as in EXAMPLE 1-2,whereby existence of the cholesterol dimer (Rf=0.65) represented by theformula (5a) was not recognized. Further, the acetone-purified productwas analyzed by ¹H-NMR and the content of the cholesterol dimer wascalculated from the proton ratio. From the result, existence of thecholesterol dimer was not recognized. Therefore the content of thecholesterol dimer represented by the formula (5a) in theacetone-purified product is 0% by weight.

[0150]¹H-NMR spectrogram of the mannan-cholesterol derivative (CHM)obtained in the manner described above is shown in FIG. 4. Theproportion of introduction of the cholesteryl group into mannan in thiscompound is calculated in the same way as in EXAMPLE 1-2. From theresult, it is found that the proportion of substitution with cholesterylgroup is 0.8 group per 100 rmonosaccharide units.

[0151] Then, the acetone-purified product obtained as above was furtherpurified by an ultracentrifugation in the same manner as in EXAMPLE 1-3to remove the lower molecular weight mannan (unsubstituted mannan). Theresults are given in Table 4. TABLE 4 Content of CHM in acetone-purified87 product (wt. %) Content of CHM in ultracentrifug.- 100 purifiedproduct (wt. %) Content of unsubstituted mannan 0 (wt. %) Content ofcholesterol dimer (wt. %) 0

EXAMPLES 3 to 6 Production of High Purity Polysaccharide ContainingSteryl Group

[0152] Using each a polysaccharide of natural origin, namely, xyloglucan(EXAMPLE 3), amylose (EXAMPLE 4), dextran (EXAMPLE 5) and a syntheticpolysaccharide, i.e. hydroxyethyl cellulose (EXAMPLE 6), each a highpurity polysaccharide-cholesterol derivative was obtained by similarprocedures for reaction as those in EXAMPLES 1-2 and 1-3.

[0153] The proportion of introduction of cholesteryl group, the rate ofremoval of the cholesterol dimer (weight %) and the content thereof(weight %) were estimated in accordance with corresponding analyticaltechniques. The content of the unsubstituted polysaccharide was alsodetermined each before and after the ultracentrifugation. The resultsare summarized in Table 5. TABLE 5 Example 3 4 5 6 Startingpolysaccharide Xyloglucan Amylose Dextran Hydroxyet. cellulose Number ofcholesteryl groups in the product¹⁾ 1.2 0.8 1.3 1.0 Purity of theacetone purified product (wt. %) 90 82 88 92 Purity ofultracentr.-purified product (wt. %) 100 100 100 100 Impurity Content ofcholesterol dimer (wt. %) 0 0 0 0 Rate of removal of cholesterol dimer(wt. %) 98.4 98.8 98.6 98.4 Content of unreacted polysacoharide (wt. %)before ultracentrifugation 10 18 12 8 after ultracentrifugation 0 0 0 0

[0154] From Table 5, it is seen that cholesteryl group can be introducedin a similar way into other polysaccharides than pullulan and mannan.

EXAMPLE 7-1 Synthesis of N-(6-isocyanatohexyl)stearyl Carbamate(Synthesis of Stearylpullulan)

[0155] In an eggplant type flask of 1 liter capacity, there are charged3.48 g (12.9 mmol) of stearyl alcohol and thereto were added 50 ml oftoluene to dissolve it, whereto 2.04 g (25.8 mmol) of pyridine werefurther added. To this mixture, there were added 30 g (178 mmol, 14.8eq.) of hexamethylene diisocyanate dissolved in 50 ml of toluene and theresulting mixture was subjected to reaction at 80° C. under a nitrogenatmosphere for about 3 hours. After termination of the reaction, tolueneand the excess of hexamethylene diisocyanate were removed under areduced pressure, whereby a pale yellow crystals were formed. Thecrystals were taken out and introduced into about one liter of hexaneand the mixture was shaken vigorously, whereupon the supernatant wasremoved by decantation. This washing procedure was repeated four times,whereupon the product was dried under a reduced pressure for three hoursat room temperature, whereby 2.75 g of a white solid product (crystals)were obtained (yield: 48.7%).

[0156] The result of analysis of this product by ¹H-NMR was as givenbelow.

[0157]¹H-NMR: δ ppm, in CDCl₃, TMS 0.88 (t, d=6.8 Hz, 3H) 1.10-1.65 (m,40H) 3.14-3.18 (m, 2H) 3.29 (t, J=6.6 Hz, 2H) 4.01-4.06 (m, 2H) 4.61 (m,1H)

[0158] From these data, it was confirmed that the product obtained isN-(6-isocyanatohexyl)stearyl carbamate represented by the followingformula (8):

[0159] On the other hand, the above product purified with acetone wereanalyzed using Preparative TLC in the same manner as in EXAMPLE 1-1,whereby existence of a stearyl dimer (Rf=0.68) represented by thefollowing formula (9) was recognized. Further, the band for the stearyldimer on the TLC was extracted with acetone and analysed quantitatively,whereby it was confirmed that the stearyl dimer was present in the whitecrystals in an amount of 3% by weight.

EXAMPLE 7-2 Synthesis of Stearyl-pullulan Derivative (STP)

[0160] In an eggplant type flask of 100 ml capacity, there are charged2.0 g (12.3 mmol as anhydrous glucose unit) of pullulan (Mw=108,000) andthereto were added 30 ml of dimethyl sulfoxide and the resulting mixturewas agitated at 80° C. under a nitrogen atmosphere to dissolve it. Tothis mixture, a solution of 70 mg (0.148 mmol) ofN-(6-isocyanatohexyl)stearyl carbamate synthesized in EXAMPLE 7-1dissolved in 1.47 g (14.6 mmol, 1.2 eq.) of pyridine was added and themixture was subjected to reaction at 90° C. for 2 hours.

[0161] After termiantion of the reaction, dimethyl sulfoxide wasdistilled off under a reduced pressure and the resulting oily residuewas dropped into 300 ml of acetone to form a precipitate to purify theproduct. After removal of the supernatant, 200 ml of acetone were addedto the resulting precipitate and the mixture was stood still overnightat room temperature. The precipitate was collected by filtration and wasdried under a reduced pressure. The so-obtained solids were dissolved indimethyl sulfoxide and the solution was charged in a dialysis bag(Spectra/Por3, a product of the firm Spectropor, an exclusion molecularweight of 3,500) and was subjected to a dialysis against distilled waterfor one week. 150 ml of the resulting polymer solution were treated byfreeze-drying in an ordinary manner, whereby 1.60 g of a white solidmatter (in the following, denoted occasionally as “acetone-purifiedproduct”) were obtained (yield 79.2%).

[0162] The results of analyses of this acetone-purified product by¹H-NMR and IR were as given below.

[0163]¹H-NMR: δ ppm, DMSO-d₆/D₂O=20/1 (vol.) 0.86-1.70 2.60-4.604.60-5.30 IR (KBr, cm⁻¹): 1680, 1180-900

[0164] From these data, it was confirmed that the product obtained is astearyl-pullulan derivative (abbreviated hereinafter sometimes as STP)represented by the following formula (10):

[0165] On the other hand, the above product purified with acetone wereanalyzed using Preparative TLC in the same manner as in EXAMPLE 1-2,whereby existence of the stearyl dimer represented by the formula (9)was not recognized. Further, the acetone-purified product was analysedby ¹H-NMR and the content of the stearyl dimer was calculated from theproton ratio. From the result, existence of the stearyl dimer was notrecognized. Therefore the content of the stearyl dimer represented bythe formula (9) in the acetone-purified product is 0% by weight.

[0166] From the integration value of the peak area of the ¹H-NMRspectrogram, the proportion of introduction of the stearyl groups intopullulan is calculated by the following calculation equation (B):

(100+2x)/43x=b/a  (B)

[0167] in which the symbols denote:

[0168] a: the peak area of the stearyl group (δ=0.86-1.70)

[0169] b: the peak area of pullulan (δ=4.60-5.30)

[0170] x: proportion of substitution with stearyl group per 100monosaccharide units.

[0171] From the calculation, it is found that the proportion ofsubstitution with stearyl group in the stearyl-pullulan derivative is0.8 group per 100 monosaccharide units.

[0172] The acetone-purified product obtained as above was furtherpurified by an ultracentrifugation as in EXAMPLE 1-3 to remove the lowermolecular weight pullulan (unsubstituted pullulan). Results are given inTable 6. TABLE 6 Content of STP in acetone-purified 92 product (wt. %)Content of STP in ultracentrifug.- 100 purified product (wt. %) Contentof unsubstituted pullulan 0 (wt. %) Content of stearyl dimer (wt. %) 0

EXAMPLES 8-1 Purification with Aprotic Polar Solvent

[0173] 10 grams of the pullulan-cholesterol derivative obtained inEXAMPLE 1-2 were dissolved in 70 grams of an aprotic polar solventdimethyl silfoxide (DMSO), whereto were added 1,400 grams of water andthe mixture was agitated using a magnetic stirrer for 10 minutes. Afterthe agitation, the mixture was stood still as such at room temperaturefor one hour. The supernatant was removed by decantation, whereupon thesame amount, as extracted, of water was added to the residue and theresulting mixture was agitated for 10 minutes using a magnetic stirrer,followed by a pause of still stand of one hour. This procedure wasrepeated twice to effect purification. Then, the lower layer was mixedwith 1,000 grams of water, whereupon the mixture was subjected to afreeze-drying over a period of two days. As the result, 7.5 grams of awhite solid matter were obtained (yield=75%). The results are summarizedin Tables 7 and 8.

[0174] By adding water to the resulting white solid matter, a 0.2 wt. %aqueous solution was prepared, which was subjected to a ultrasonicationusing a sonicatore of probe type (TOMY of the firm URP, with a probeouter diameter of 5 mm, at 40 W) for 15 minutes. A sample collectedafter the ultrasonication was examined by a size exclusionchromatography (SEC) under the conditions given below. The results areshown in FIG. 5. No peak is found in FIG. 5 and, thus, it is seen that apullulan-cholesterol derivative of high purity in which unsubstitutedpullulan had been removed was obtained.

[0175] ∘ Conditions in the SEC analysis: Apparatus used: TOSOH HPSECSYSTEM (trademark, of Tosoh K. K. ) Column: TSK-gel G4000SWXL(trademark, of Tosoh K. K. ) Eluent: 0.02% NaN₃ in deionized water Flowrate: 0.5 ml/min. Temperature: 35° C. Detector: RI (differentialrefractometer)

EXAMPLES 8-2 to 8-6

[0176] Purification of each product was conducted using each of thehydrophobic group-containing polysaccharide and the aprotic polarsolvent given in Tables 7 and 8 under the purification conditions givenin Tables 7 and 6 by the same operation as in EXAMPLE 8-1. In all thepurification operations, the procedure of phase separation into twolayers was repeated twice. The results are summarized in Tables 7 and 8.

[0177] By SEC analysis of each of the hydrophobic group-containingpolyasccharides, no peak for the unsubstituted polysaccharide was foundin all the EXAMPLES and, thus, it was confirmed that all the products ofhydrophobic group-containing polysaccharide had been purified to nearly100 weight % purity. TABLE 7 Results of Acetone-Purified Product Example8-1 8-2 8-3 8-4 8-5 8-6 Starting polysaccharide Pullulan PullulanPullulan Pullulan Mannan Pullulan Hydrophobic group Cholester.Cholester. Cholester. Cholester. Cholester. Stearyl Introduct.proportion¹⁾ 1.1 1.3 2.9 1.1 1.9 0.8 of hydrophobic group Product CHPCHP CHP CHP CHM STP Content of unreacted 5 10 16 5 20 16 polysaccharide(wt. %) Content of dimer (wt. %) 0 0 0 0 0 0 Purity (wt. %) 95 90 84 9580 84

[0178] TABLE 8 Purification with Aprotic Polar Solvent and the ResultsExample 8-1 8-2 8-3 8-4 8-5 8-6 Amount of Acetone - (g) 10 40 5 10 2 3purified product Aprotic polar solvent Kind¹⁾ DMSO DMSO DMSO DMF DMSODMAc Amount used (g) 70 320 50 70 12 18 Times weight 7 8 10 7 6 6 Amountof water used (g) 1,400 5,000 800 1,000 150 300 Treating proceduresWater wash Water wash Water wash Water wash Water wash Water wash Twolayer- Two layer- Two layer- Two layer- Two layer- Two layer- separationseparation separation separation separation separation Two steps Twosteps Two steps Two steps Two steps Two steps Treating time (hr) 5 5 4 54 4 Yield (wt. %) 75 75 80 72 65 69 Purity (wt. %) 100 100 100 100 100100 Content of unreacted 0 0 0 0 0 0 polysaccharide (wt. %)

[0179] In the purification using an aprotic polar solvent, a though-putof purification of 10 grams in about 5 hours was able to be attained andthe yield was also superior as it amounted to as high as 65% by weightor higher. The purification for each product was over within about 2hours. There is no limitation in principle for the amount to be purifiedand mass purification can be realized easily. From these points of view,it is seen that the purification with an aprotic polar solvent is veryeffective for industrial mass production.

INDUSTRIAL APPLICABILITY

[0180] The product of polysaccharide containing hydrophobic groupobtained by the production process according to the present inventioncan be utilized as a coating material for coating drug carrierscontaining encapsulated therein drugs. For example, the product can beused as the coating material for coating drug carriers, such as liposomemicrcapsules, microspheres, O/W emulsions and erythrocyte ghost. In thiscase, the high purity polysaccharide containing hydrophobic groupaccording to the present invention can be used safely as the medicalmaterial, since it is present as a high purity product having scarcecontents of by-products and of the unsubstituted polysaccharide.

1. A process for producing a high purity polysaccharide containinghydrophobic group, comprising a first process step of producing anisocyanate group-containing hydrophobic compound, wherein one mole of ahydroxyl group-containing hydrocarbon having 12-50 carbon atoms or of asterol is reacted with a diisocyanate represented by OCN—R¹—NCO in whichR¹ is a hydrocarbyl of 1-50 carbon atoms and a second process step ofproducing the polysaccharide containing hydrophobic group composed ofthe hydrocarbon group of 12-50 carbon atoms or of the steryl group,wherein the isocyanate group-containing hydrophobic compound obtained inthe first process step is reacted with one or more polysaccharides,wherein the reaction product in the second process step is purifiedusing a solvent based on ketone.
 2. The process as claimed in claim 1,wherein the polysaccharide is selected from the group consisting ofpullulan, amylopectin, amylose, dextran, hydroxyethyl cellulose,hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan,xyloglucan and water-soluble cellulose.
 3. The process as claimed inclaim 1 or 2, wherein the solvent based on ketone comprises one or moreselected from the group consisting of acetone, methyl ethyl ketone,diethyl ketone and diisopropyl ketone.
 4. The process as claimed in anyone of claims 1 to 3, wherein the hydrophobic group-containingpolysaccharide has a group represented by —XH in which X is oxygen atomor a nitrogen-containing group represented by NY with Y being hydrogenatom or a hydrocarbyl of 1-10 carbon atoms wherein 0.1-10 —XH groups per100 monosaccharide units constituting the polysaccharide are replaced byone or more hydrophobic groups represented by the formula (1), namely,

in which X is the same as given above, R¹ denotes a hydrocarbyl having1-50 carbon atoms and R² denotes a hydrocarbon group of 12-50 carbonatoms or a steryl group.
 5. The process as claimed in claim 4, whereinR² in the formula (1) denotes a steryl group.
 6. The process as claimedin any one of claims 1 to 5, wherein the content of the hydrophobicgroup-containing polysaccharide in the product purified using thesolvent based on ketone is as high as 80% by weight or more.
 7. Theprocess as claimed in claim 6, wherein the content of unsubstitutedpolysaccharide is as low as 20% by weight or less.
 8. The process asclaimed in claim 6 or 7, wherein the product has a content of theimpurity product, in which both the two NCO groups in the diisocyanateare reacted with the hydroxyl group-containing hydrocarbon having 12-50carbon atoms or with the sterol, as low as 0.05% by weight or less. 9.The process as claimed in any one of claims 1 to 8, wherein the productpurified using a solvent based on ketone is subjected to a furtherpurification by dispersing the product finely in water under anultrasonic treatment, with subsequent ultracentrifugal separation. 10.The process as claimed in claim 9, wherein the content of thehydrophobic group-containing polysaccharide in the purified product fromthe ultracentrifugal separation is as high as 98% by weight or more. 11.The process as claimed in claim 10, wherein the content of unsubstitutedpolysaccharide is as low as 2% by weight or less.
 12. The process asclaimed in claim 10 or 11, wherein the content of the impurity product,in which both the two NCO groups in the diisocyanate are reacted withthe hydroxyl group-containing hydrocarbon having 12-50 carbon atoms orwith the sterol, is as low as 0.05% by weight or less.
 13. The processas claimed in any one of claims 1 to 8, wherein the product purifiedusing the solvent based on ketone is further subjected to a purificationprocedures comprising dissolving the product in an aprotic polarsolvent, admixing water to the resulting solution to cause theunsubstituted polysaccharide to be transferred to the aqueous phase andremoving the aqueous phase separated by phase separation.
 14. Theprocess as claimed in claim 13, wherein the further purification of theproduct purified using the solvent based on ketone is performed bydissolving the product in the aprotic polar solvent of an amount of 3-50times the weight of the product and admixing water to the resultingsolution in an amount of at least 5 times the weight of the solution.15. The process as claimed in claim 13 or 14, wherein the aprotic polarsolvent comprises one or more selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide. 16.The process as claimed in any one of claims 13 to 15, wherein thecontent of the hydrophobic group-containing polysaccharide in thepurified product purified using the aprotic polar solvent is as high as98% by weight or more.
 17. The process as claimed in claim 16, whereinthe content of the unsubstituted polysaccharide is as low as 2% byweight or less.
 18. The process as claimed in claim 16 or 17, whereinthe content of the impurity product, in which both the two NCO groups inthe diisocyanate are reacted with the hydroxyl group-containinghydrocarbon having 12-50 carbon atoms or with the sterol, is as low as0.02% by weight or less.
 19. A high purity product of polysaccharidecontaining hydrophobic group which contains at least 80% by weight ofthe polysaccharide containing hydrophobic group, wherein thepolysaccharide is one having a group represented by —XH in which X isoxygen atom or a nitrogen-containing group represented by NY with Ybeing hydrogen atom or a hydrocarbyl of 1-10 carbon atoms wherein 0.1-10—XH groups per 100 monosaccharide units constituting the polysaccharideare replaced by one or more hydrophobic groups represented by theformula (1), namely,

in which X is the same as given above, R¹ denotes a hydrocarbyl having1-50 carbon atoms and R² denotes a hydrocarbon group of 12-50 carbonatoms or a steryl group, the said polysaccharide containing hydrophobicgroup being obtained by a process comprising a first process step ofproducing an isocyanate group-containing hydrophobic compound, whereinone mole of a hydroxyl group-containing hydrocarbon having 12-50 carbonatoms or of a sterol is reacted with a diisocyanate represented byOCN—R¹—NCO in which R¹ is a hydrocarbyl of 1-50 carbon atoms, a secondprocess step of producing the polysaccharide containing hydrophobicgroup composed of the hydrocarbon group of 12-50 carbon atoms or of thesteryl group, wherein the isocyanate group-containing hydrophobiccompound obtained in the first process step is reacted with one or morepolysaccharide, and purifying the reaction product from the secondprocess step using a solvent based on ketone.
 20. The high purityproduct of polysaccharide containing hydrophobic group as claimed inclaim 19, wherein the polysaccharide is selected from the groupconsisting of pullulan, amylopectin, amylose, dextran, hydroxyethylcellulose, hydroxyethyl dextran, mannan, levan, inulin, chitin,chitosan, xyloglucan and water-soluble cellulose.
 21. The high purityproduct of polysaccharide containing hydrophobic group as claimed inclaim 19 or 20, wherein R² in the formula (1) is steryl.
 22. The highpurity product of polysaccharide containing hydrophobic group as claimedin any one of claims 19 to 21, wherein the content of unsubstitutedpolysaccharide is as low as 20% by weight or less.
 23. The high purityproduct of polysaccharide containing hydrophobic group as claimed in anyone of claims 19 to 22, wherein the content of the impurity product, inwhich both the two NCO groups in the diisocyanate are reacted with thehydroxyl group-containing hydrocarbon having 12-50 carbon atoms or withthe sterol, is as low as 0.05% by weight or less.
 24. The high purityproduct of polysaccharide containing hydrophobic group as claimed in anyone of claims 19 to 23, wherein the product purified using the solventbased on ketone is subjected to a further purification by dispersing theproduct finly in water under an ultrasonic treatment, with subsequentultracentrifugal separation.
 25. The high purity product ofpolysaccharide containing hydrophobic group as claimed in any one ofclaims 19 to 23, obtained by subjecting the product purified using thesolvent based on ketone to a further purification procedures comprisingdissolving the product in an aprotic polar solvent, admixing water tothe resulting solution to cause the unsubstituted polysaccharide to betransferred to the aqueous phase and removing the aqueous phaseseparated by phase separation.